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Detergent-Solubilized Membrane Protein Reconstituted into Nanodiscs

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Creative Biolabs provides membrane protein solubilization services using nanodiscs system, such as reconstitution of G-protein coupled receptors (GPCRs) into self-assembling nanodiscs.

Nanodisc technology can offer near-native membrane environment compared to micelles, bicelles, and other detergent-lipid mixed system. Thus, nanodiscs are widely used for purifying seven-transmembrane GPCRs in a soluble and functional state.

Figure 1. Assembly of β2-adrenergic receptor (β2AR)-Nanodiscs. (Biotechniques, 2006)

Reconstitution of GPCRs into nanodiscs provides a wide series of advantages. The use of nanodiscs does not lead to high concentrations of freely diffusing lipids or detergent molecules, and the nanodisc structure is compact. These features permit relatively high concentrations of protein per volume, enabling bulk spectroscopic measurements. Furthermore, in the case of the nanodisc the presence of MSPs permits additional labeling for spectroscopic analysis. MSPs can be expressed in E. coli, a system that allows genetic incorporation of a variety of unnatural amino acids, including fluorescent ones, by stop-codon suppression. However, nanodiscs also have some advantages: Firstly, nanodiscs cannot in principle reproduce the difference between the extracellular and intracellular environments — something that vesicles can do. Secondly, nanodiscs is the strict limit on the diameter of the particle.

The β2-adrenergic receptor (β2AR) was the first GPCR cloned and represents a GPCR that has been extensively investigated as a major pharmaceutical target for the control of asthma and hypertension. Creative Biolabs can provide Mempro™ cell-based protein production (such as β2AR or other G-proteins), reconstitution of β2AR with MSP and the lipids at ratios designed to result in an excess of target-free nanodiscs. The large excess of scaffold protein and lipids compared with β2AR is added to minimize nonspecific receptor loss. During this step, appropriate detergent is added to the solution followed by β2AR solubilization. Final concentrations of detergent are typically greater than 12 mM and well above the 4 mM minimum lipid concentration determined for efficient formation of nanodisc complexes (Figure 1). The reconstitution mixtures are incubated followed by addition of Bio-Beads to initiate the self-assembly process by removal of detergents, and the Bio-Beads are removed by centrifugation. Then, the reconstitution mixtures are concentrated and purified through utilization of an engineered C-terminal FLAG affinity tag on the receptor, and empty nanodiscs can be removed from the size-purified nanodisc mixture. Finally, the purified β2AR-nanodisc mixtures can be used immediately for further projects.

Creative Biolabs can provide other membrane protein production services. Please feel free to contact us for a detailed quote.

References:
E. Serebryany, et al. (2012). Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors. Biochim. Biophys. Acta. 1818(2): 225-233.
J. Leitz, et al. (2006). Functional reconstitution of beta2-adrenergic receptors utilizing self-assembling nanodisc technology. Bio.Techniques, 40(5): 601-612.




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